DESCRIPTION (verbatim from applicant's abstract): Ubiquitous kinesin is a microtubule-based motor protein that moves membrane-bounded organelles in fast axonal transport, Golgi to ER transport, and the exocytic pathway. It also functions in transport of non-membranous vimentin particles and organization of the intermediate filaments. Loss of kinesin function is embryonic lethal in most organisms, and causes disruptions in axonal transport similar to those observed in amyotrophic lateral sclerosis. Notably, kinesin can move long distances along microtubules as a single, isolated molecule (termed processive movement). Recent work has allowed this motion to be visualized using the new method of single molecule fluorescence microscopy, and has also shown that the kinesin family motors Ncd, unc104, and KRP8.5/95 are far less processive than ubiquitous kinesin. Ncd functions in the assembly and function of mitotic and meiotic spindles, unc104 functions in axonal vesicle transport, and KRP85/95 functions in membrane transport and the assembly of motile and sensory cilia. Kinesin's ability to move long distances has been thought to be related to its role in membrane transport, but the low or nonexistent processivity of uncl04 and KRP85/95 calls into question our understanding of why ubiquitous kinesin has this property, and hints that it may be related to another biological function. The research proposed here is aimed at biophysical tests of the mechanism by which ubiquitous kinesin accomplishes long&#8209;distance movement, and cell biological tests of what role this ability plays in kinesin's functions in vivo. To clarify these issues, we will 1) Use novel single molecule imaging methods to detect how the parts of kinesin move in relation to one another during motility, 2) Determine which parts of kinesin account for its unique properties, 3) Determine how ATP hydrolysis and movement are related, and how this relationship is affected when one of kinesin's two chemomechanical domains is absent, and 4) dynamically image kinesin as it moves in living cells, and use genetic approaches in C. elegans to determine how kinesin's in vivo movements and cellular functions are affected when it is altered so as to render it incapable of long distance single molecule movement, without otherwise affecting its motor function. These experiments should be of great value in understanding the kinesin motor mechanism, and in illuminating general principles of how the biophysical properties of motor proteins are related to their biological functions in the cell.